Vector

A vector, such as a plasmid, yeast, or animal virus genome, used to introduce

foreign genetic material into a host cell in order to replicate and amplify the foreign DNA
sequences as a recombinant molecule.

Any vector designed to enable the expression of a cloned gene. Vectors for
expression in Escherichia coli may use the lac promoter, for example. Vectors for
expression in mammalian cells are more complex; they may have the CMV
(cytomegalovirus) promoter, which is active in a wide range of cell types, and other
features such as termination sequences and sometimes an intron.

In molecular biology, a vector is a DNA molecule used as a vehicle to transfer

foreign genetic material into another cell. The four major types of vectors are plasmids,
bacteriophages and other viruses, cosmids, and artificial chromosomes. Common to all
engineered vectors are an origin of replication, a multicloning site, and a selectable
marker.

The vector itself is generally a DNA sequence that consists of an insert

(transgene) and a larger sequence that serves as the "backbone" of the vector. The
purpose of a vector which transfers genetic information to another cell is typically to
isolate, multiply, or express the insert in the target cell.

Vectors called expression vectors (expression constructs) specifically are for the
expression of the transgene in the target cell, and generally have a promoter sequence that
drives expression of the transgene. Simpler vectors called transcription vectors are only
capable of being transcribed but not translated: they can be replicated in a target cell but
not expressed, unlike expression vectors. Transcription vectors are used to amplify their
insert.

Insertion of a vector into the target cell is usually called transformation for
bacterial cells, transfection for eukaryotic cells, although insertion of a viral vector is
often called transduction.

Characteristics
Two common vectors are plasmids and viral vectors.

Plasmids

Plasmids are double-stranded generally circular DNA sequences that are capable
of automatically replicating in a host cell. Plasmid vectors minimalistically consist of an
origin of replication that allows for independent replication of the plasmid in the host and
also the transgene insert.

Modern plasmids generally have many more features, notably including a

"multiple cloning site" which includes nucleotide overhangs for insertion of an insert, and
multiple restriction enzyme consensus sites to either side of the insert.
 In the case of plasmids utilized as transcription vectors, incubating bacteria with
plasmids generates hundreds or thousands of copies of the vector within the bacteria in
hours, and the vectors can be extracted from the bacteria, and the multiple cloning site
can be cut by restriction enzymes to excise the hundredfold or thousandfold amplified
insert.

These plasmid transcription vectors characteristically lack crucial sequences that

code for polyadenylation sequences and translation termination sequences in translated
mRNAs, making protein expression from transcription vectors impossible.

Plasmids may be- conjugative/transmissible and non conjugative. conjugative-

mediate DNA transfer through conjugation and therefore spread rapidly among the
bacterial cells of a population. e.g- F plasmid, many R and some col plasmids.
nonconjugative- do not mediate DNA through conjugation. e.g.- many R and col plasmids

Viral vectors

Viral vectors are generally genetically-engineered viruses carrying modified viral

DNA or RNA that has been rendered noninfectious, but still contain viral promoters and
also the transgene, thus allowing for translation of the transgene through a viral promoter.

However, because viral vectors frequently are lacking infectious sequences, they
require helper viruses or packaging lines for large-scale transfection.

Viral vectors are often designed for permanent incorporation of the insert into the
host genome, and thus leave distinct genetic markers in the host genome after
incorporating the transgene. For example, retroviruses leave a characteristic retroviral
integration pattern after insertion that is detectable and indicates that the viral vector has
incorporated into the host genome.

Transcription is a necessary component in all vectors: the premise of a vector is

to multiply the insert (although expression vectors later also drive the translation of the
multiplied insert). Thus, even stable expression is determined by stable transcription,
which generally depends on promoters in the vector. However, expression vectors have a
variety of expression patterns: constitutive (consistent expression) or inducible
(expression only under certain conditions or chemicals). This expression is based on
different promoter activities, not post-transcriptional activities. Thus, these two different
types of expression vectors depend on different types of promoters.

Viral promoters are often used for constitutive expression in plasmids and in viral
vectors because they normally reliably force constant transcription in many cell lines and
types.

Inducible expression depends on promoters that respond to the induction

conditions: for example, the murine mammary tumor virus promoter only initiates
transcription after dexamethasone application and the Drosophilia heat shock promoter
only initiates after high temperatures. Transcription is the synthesis of mRNA. Genetic
information is copied from DNA to RNA
Expression
Expression vectors require translation of the vector's insert, thus requiring more
components than simpler transcription-only vectors. Expression vectors require sequences
that encode for:

• Polyadenylation tail: Creates a polyadenylation tail at the end of the transcribed

pre-mRNA that protects the mRNA from exonucleases and ensures transcriptional
and translational termination: stabilizes mRNA production.
• Minimal UTR length: UTRs contain specific characteristics that may impede
transcription or translation, and thus the shortest UTRs or none at all are encoded
for in optimal expression vectors.
• Kozak sequence: Vectors should encode for a Kozak sequence in the mRNA,
which assembles the ribosome for translation of the mRNA.

(Above conditions are necessary for Expression vectors in eukaryotes, not prokaryotes)

Features
Modern vectors may encompass additional features besides the transgene insert and a
backbone:

• Promoter: Necessary component for all vectors: used to drive transcription of the
vector's transgene.
• Genetic markers: Genetic markers for viral vectors allow for confirmation that
the vector has integrated with the host genomic DNA.
• Antibiotic resistance: Vectors with antibiotic-resistance open reading frames
allow for survival of cells that have taken up the vector in growth media
containing antibiotics through antibiotic selection.
• Epitope: Vector contains a sequence for a specific epitope that is incorporated
into the expressed protein. Allows for antibody identification of cells expressing
the target protein.
• β-galactosidase: Some vectors contain a sequence for β-galactosidase, an enzyme
that digests galactose, within which a multiple cloning site, the region in which a
gene may be inserted, is located. An insert successfully ligated into the vector will
disrupt the β-galactosidase gene and disable galactose digestion. Cells containing
vector with an insert may be identified using blue/white selection by growing cells
in media containing an analogue of galactose (X-gal). Cells expressing β-
galactosidase (therefore doesn't contain an insert) appear as blue colonies. White
colonies would be selected as those that may contain an insert. Other proteins
which may function similarly as a reporter include green fluorescent protein and
luciferase.
• Targeting sequence: Expression vectors may include encoding for a targeting
sequence in the finished protein that directs the expressed protein to a specific
organelle in the cell or specific location such as the periplasmic space of bacteria.
• Protein purification tags: Some expression vectors include proteins or peptide
sequences that allows for easier purification of the expressed protein. Examples
include polyhistidine-tag, glutathione-S-transferase, and maltose binding protein.
Some of these tags may also allow for increased solubility of the target protein.
 The target protein is fused to the protein tag, but a protease cleavage site
positioned in the polypeptide linker region between the protein and the tag allows
the tag to be removed later.

Expression vector
An expression vector, otherwise known as an expression construct, is generally
a plasmid that is used to introduce a specific gene into a target cell. Once the expression
vector is inside the cell, the protein that is encoded by the gene is produced by the
cellular-transcription and translation machinery ribosomal complexes.

The plasmid is frequently engineered to contain regulatory sequences that act as

enhancer and promoter regions and lead to efficient transcription of the gene carried on
the expression vector.[1]

The goal of a well-designed expression vector is the production of large amounts

of stable messenger RNA, and therefore proteins. Expression vectors are basic tools for
biotechnology and the production of proteins such as insulin that are important for
medical treatments of specific diseases like diabetes.

After expression of the gene product, the purification of the protein is required;
but since the vector is introduced to a host cell, the protein of interest should be purified
from the proteins of the host cell. Therefore, to make the purification process easy, the
cloned gene should have a tag. This tag could be histidine (His) tag or any other marker
peptide.

Expression vectors are used for molecular biology techniques such as site-
directed mutagenesis. Cloning vectors, which are very similar to expression vectors,
involve the same process of introducing a new gene into a plasmid, but the plasmid is
then added into bacteria for replication purposes. In general, DNA vectors that are used in
many molecular-biology gene-cloning experiments need not result in the expression of a
protein.

Expression vectors must have expression signals such as a strong promoter, a

strong termination codon, adjustment of the distance between the promoter and the cloned
gene, and the insertion of a transcription termination sequence and a PTIS (portable
translation initiation sequence).

Some Common Uses

In recent years, expression vectors have been used to introduce specific genes in
organisms, especially plants used in agriculture.

Expression vectors have been used to introduce a vitamin A precursor, beta-

carotene, into rice plants. This product is called golden rice.
 This process has also been used to introduce a gene into plants that produces an
insecticide, called Bacillus thuringiensis toxin or Bt toxin which reduces the need for
farmers to apply insecticides since it is produced by the modified organism.

In addition expression vectors are used to extend the ripeness of tomatoes by

altering the plant so that it produces less of the chemical that causes the tomatoes to rot.[2]

There has been controversy over using expression vectors to modify crops due to
the fact that there are unknown health risks, possibilities of companies patenting certain
crops, and ethical concerns. Nevertheless, this technique is still being used and heavily
researched. eg. baculovirus is commonly used as expression vectors for insect cells

Shuttle vector
A shuttle vector is a vector (usually a plasmid) constructed so that it can
propagate in two different host species [1]. Therefore, DNA inserted into a shuttle vector
can be tested or manipulated in two different cell types. The main advantage of these
vectors is they can be manipulated in E. coli then used in a system which is more difficult
or slower to use (e.g. yeast, other bacteria).

Shuttle vectors include plasmids that can propagate in eukaryotes and prokaryotes
(e.g. both Saccharomyces cerevisiae and Escherichia coli) or in different species of
bacteria (e.g. both E. coli and Rhodococcus erythropolis).

There are also adenovirus shuttle vectors, which can propagate in E. coli and
mammals.

Shuttle vectors are frequently used to quickly make multiple copies of the gene in
E. coli (amplification). They can also be used for in vitro experiments and modifications
(e.g. mutagenesis, PCR)

One of the most common types of shuttle vectors is the yeast shuttle vector [2].
Almost all commonly used S. cerevisiae vectors are shuttle vectors. Yeast shuttle vectors
have components that allow for replication and selection in both E. coli cells and yeast
cells. The E. coli component of a yeast shuttle vector includes an origin of replication and
a selectable marker, e.g. antibiotic resistance, Beta lactamase. The yeast component of a
yeast shuttle vector includes an autonomously replicating sequence (ARS), a yeast
centromere (CEN), and a yeast selectable marker (e.g. URA3, a gene that encodes an
enzyme for uracil synthesis).